ORIGINAL ARTICLE

Cortical -Aminobutyric Acid Type ABenzodiazepine Receptors in Recovery From Alcohol Dependence
Relationship to Features of Alcohol Dependence and Cigarette Smoking
Julie K. Staley, PhD; Christopher Gottschalk, MD; Ismene L. Petrakis, MD; Ralitza Gueorguieva, PhD; Stephanie OMalley, PhD; Ronald Baldwin, PhD; Peter Jatlow, PhD; Nicolaas Paul L. G. Verhoeff, MD, PhD; Edward Perry, MD; David Weinzimmer, BA; Erin Frohlich, BA; Elizabeth Ruff, BA; Christopher H. van Dyck, MD; John P. Seibyl, MD; Robert B. Innis, MD, PhD; John H. Krystal, MD

Context: Adaptations in -aminobutyric acid type A (GABAA)benzodiazepine receptors contribute to the neurobiology of human alcohol dependence and withdrawal. Objective: To study GABAA-benzodiazepine receptor adaptations in subjects with alcohol dependence over the first month of sobriety. Design: Inpatients who were not receiving medication, were either smokers or nonsmokers, and had alcohol dependence completed 2 iodine I 123labeled iomazenil single-photon emission computed tomographic scans: 1 scan at a meanSD of 4.92.5 days of sobriety (n=23) and 1 scan at a meanSD of 29.87.6 days of sobriety (n=20). Participants in a matched group of healthy subjects (n=15) completed 1 single-photon emission computed tomographic scan. Participants: Men with alcohol dependence (n=27) and a matched healthy comparison group (n = 15). Main Outcome Measures : 123I-iomazenil singlephoton emission computed tomographic images were converted to units of distribution volume (regional activity/ free 123I-iomazenil) and were analyzed using voxel-based statistical parametric mapping and regions of interest analyses. The relationships between 123I-iomazenil dis-

tribution volume, clinical features of alcohol dependence, and smoking status were evaluated.
Results: 123I-iomazenil uptake was elevated in several cor-

tical regions, with a more prominent increase in nonsmokers with alcohol dependence as compared with smokers with alcohol dependence at 1 week of abstinence from alcohol. No significant differences were observed at 4 weeks of abstinence. At 1 week of abstinence, frontal 123I-iomazenil uptake correlated with the severity of alcohol withdrawal and the number of days since the last alcoholic drink was consumed. No significant associations were observed for smokers with alcohol dependence.
Conclusions: These data demonstrate time-dependent regulation of cortical GABAA-benzodiazepine receptors associated with the recovery from alcohol dependence. Higher GABAA-benzodiazepine receptor levels during acute withdrawal may reflect a compensation for reduced receptor function, which is thought to contribute to alcohol tolerance and withdrawal. The subsequent decline may reflect normalization of GABAA receptor function with sobriety. Smoking may attenuate GABAA receptor adaptations associated with alcohol dependence and may contribute to the comorbidity between alcoholism and smoking.

Arch Gen Psychiatry. 2005;62:877-888 the principal pharmacotherapy for alcohol withdrawal symptoms.4-6 While there is controversy regarding whether ethanol has primarily direct or indirect facilitatory effects on GABAA receptors,2,7,8 recent evidence of potent direct ethanol effects on GABAA receptor subgroups and the mapping of molecular domains of GABAA receptors through which ethanol acts9 highlight the importance of GABAA receptors for ethanol action. Ethanol and BZ enhance GABAergic function through distinct interactions with the GABAA receptor complex.10

Author Affiliations are listed at the end of this article.

plicate -aminobutyric acid type A (GABAA) receptors in the neurobiology of alcohol tolerance and dependence, including (1) facilitatory effects of ethanol on 2L-containing GABAA receptors,1 (2) generalization between the discriminative stimulus effects of ethanol and GABAA receptor agonists, (3) cross-tolerance between ethanol and benzodiazepines (BZs), and (4) the ability of BZs and other GABAA receptor agonists to suppress alcohol withdrawal.2,3 In fact, BZs are

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Neuroimaging studies of patients with alcohol dependence who have been sober for at least 1 month generally suggest that GABAA receptors are decreased. All11-14 but one15 of these studies demonstrated decreased binding of the BZ antagonists carbon 11labeled flumazenil and iodine I 123labeled iomazenil in frontal, parietal, and temporal cortices. These studies may be consistent with cerebral metabolic data suggesting that the response to lorazepam is decreased in patients who have been sober for 6 to 32 days16 and continues to decrease over 2 months of abstinence.17 However, it is possible that both the receptor-based and metabolic neuroimaging findings could reflect neurotoxic consequences of alcoholism or GABA-related disturbances that preceded the onset of alcohol dependence. Human postmortem studies report unchanged,18,19 increased,20 and decreased21,22 binding of BZ agonists in the frontal cortex, hippocampus, and cerebellum. In a small study of recently detoxified patients with alcohol dependence, 2 subjects demonstrated increased GABAA-BZ receptor availability whereas the other 3 subjects exhibited unchanged or slightly decreased GABAA-BZ receptor availability.23 When reviewed collectively with the earlier in vivo studies in subjects with alcoholism who have been abstinent for greater than 1 month, these studies suggest that levels of GABAA-BZ receptors are elevated or similar to control levels during acute abstinence and decreased with prolonged abstinence, and they also suggest that factors other than the length of abstinence may alter the levels of GABAA-BZ receptors during alcohol withdrawal. We hypothesized that some of the conflicts in the existing literature might reflect the confounding effects of time-dependent changes in GABAA-receptor populations during recovery (return to baseline) from alcohol dependence as well as other clinical features, including severity of alcohol dependence and withdrawal, subtype of alcoholism (reflected in age at onset), and the frequent comorbidity of alcohol dependence and tobacco smoking. The objectives of the current study were to directly evaluate whether GABAA-BZreceptor levels change over time during recovery from alcohol dependence, to explore the relationships to clinical correlates of alcohol dependence, and to determine if tobacco smoking modifies GABAA-BZ receptors during early sobriety.
METHODS

SUBJECTS
Thirty-six men with alcohol dependence and 15 age-matched healthy men provided written informed consent for participation in this study. The study was approved by the Department of Psychiatry Research Committee, Human Investigation Committee, and Radiation Safety Committee, Yale University School of Medicine (New Haven, Conn), and the Human Subjects Subcommittee of the West Haven Veterans Affairs Connecticut Healthcare System (West Haven, Conn). Eligibility was evaluated with a structured interview, physical examination, laboratory blood tests, urine drug screen, and an electrocardiogram. Control subjects had no history or evidence of a serious medical or neurological illness, did not meet criteria for any psychiatric or substance abuse diagnosis as determined by the Structured Clinical Interview for DSM-IV nonpatient version, had no

history of a DSM-IV Axis I psychiatric disorder in a first-degree relative, and had not used psychotropic substances other than alcohol, nicotine, or tobacco for at least 1 year preceding the study. Control subjects drank less than 8 alcoholic drinks per week as defined by the timeline follow-back (ie, 1 drink=12 oz of beer, 5 oz of wine [10% alcohol], 1.5 oz of hard liquor, etc)24 and were asked to refrain from alcoholic and caffeine-containing drinks for 2 weeks preceding the single-photon emission computed tomographic (SPECT) scan. Participants in the alcohol-dependent group met DSM-IV criteria for alcohol dependence and currently had no other Axis I disorder other than nicotine dependence, as determined by the Structured Clinical Interview for DSM-IV.25 They had no medical or neurological disorders that might compromise the interpretation of the SPECT data, had not taken any psychotropic medications in the last month, and demonstrated alcohol withdrawal symptoms of a Clinical Institute Withdrawal Assessment4 score of less than 10 at the initial assessment. Subjects with alcohol dependence were accepted with abnormal liver function test values for -glutamyltransferase, serum glutamic oxaloacetic transaminase, and serum glutamate pyruvate transaminase of up to 3 times the upper normal limit in recognition of the effect of recent drinking on liver function. Subjects with alcohol dependence were admitted into the Connecticut Mental Health Center Clinical Neuroscience Research Unit (New Haven) for 1 month. They were monitored for alcohol withdrawal symptoms using the Clinical Institute Withdrawal Assessment every 6 hours after admission until scores were consistently 0. Patients with Clinical Institute Withdrawal Assessment ratings of 10 or higher were evaluated to determine if immediate BZ administration was necessary. The BZs were automatically given if the Clinical Institute Withdrawal Assessment score exceeded 12. Once BZs were administered, no subsequent tests were performed for at least 1 month; ie, the first imaging session was cancelled and, since longterm BZ administration does not alter GABAA-BZ receptor levels,26 subjects were encouraged to participate in the second scan. Of the 36 patients who signed consent forms, 10 subjects were excluded for the following reasons: (1) withdrew consent and terminated participation before completing an imaging session; (2) did not have a magnetic resonance image taken; (3) had abnormal magnetic resonance image results; (4) had firstdegree family history of depression; (5) had cocaine use discovered after the study; or (6) there was difficulty in placing the intravenous needle and/or collecting blood samples during the SPECT scan. Three subjects with alcohol dependence participated in the second scan but did not participate in the first scan because they received BZ to treat withdrawal symptoms, the camera was not available (because of a change in location), and entrance into the study was after the first week of abstinence. Six subjects with alcohol dependence dropped out after the first scan and did not participate in the second scan. Alcohol consumption in the month preceding the SPECT scan was determined using the timeline follow-back.24 Age at onset of alcohol dependence was obtained from the Structured Clinical Interview for DSM-IV. Antisocial personality disorder was determined using the Structured Clinical Interview for DSM-IV Axis II. Severity of alcohol dependence was assessed using the Alcohol Dependence Scale,27 and alcohol craving was assessed on the day of the SPECT scan using the Tiffany Scale.28 Subjects with alcohol dependence had SPECT images taken within 10 days and/or at 4 weeks after their last alcoholic drink. Smokers in the control group and the alcohol-dependent group were instructed to maintain their normal smoking patterns. Inpatient smokers with alcohol dependence were limited to smoking 4 times per day. Smoking characteristics, including the ages at which the subjects began smoking, the total number of years they smoked, the number of cigarettes smoked per day, and the

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number of attempts to quit smoking, were documented. Nicotine dependence was evaluated using the Fagerstrom Test for Nicotine Dependence.29,30 Plasma cotinine (the principal metabolite of nicotine) was measured on the day of the SPECT scan. All nonsmokers with alcohol dependence and 8 of the 10 control nonsmokers were never smokers as defined by a lifetime history of smoking less than 40 cigarettes or nicotine-related products.

PLASMA COTININE ASSAY

Cotinine concentrations in serum were assayed using reversephase high-performance liquid chromatography. The procedure31 was modified by substitution of an aqueous microvolume back-extraction clean-up step in place of solvent evaporation. Following the addition of an internal standard (2phenylimidazole), cotinine was extracted from alkalinized serum with a 40:60 mixture of dichloromethane and hexane. Following a microvolume back-extraction into 0.1M phosphoric acid, the aqueous phase was analyzed by reverse-phase chromatography on a C6 reverse-phase column using a mobile phase of 10% acetonitrile buffered to pH 4.8 and containing 20 mL of triethylamine and 0.6 g/L of octane-sulfonic acid. Between-day coefficients of variation, in routine use at concentrations of 20 g/L and 200 g/L, were 11.6% and 6.6%, respectively.

istered to the emission image and reoriented to the intercommissural plane. Standardized 2-dimensional region of interest templates were placed on the medial frontal cortex (12.3 cm2), anterior cingulate (3.1 cm2), hippocampal-amygdala area (5.4 cm2), and cerebellum (25 cm2). Three-dimensional volumes of interest were transferred to coregistered SPECT images. Regional activities from right and left hemispheres were averaged, decay corrected, and expressed as kilobecquerels per cubic centimeter using a calibration factor of 337.44 Bq/cpm derived from a 123Idistributed source phantom. Regional activities (kilobecquerels per cubic centimeter) were normalized to free 123I-iomazenil (kilobecquerels per milliliter) plasma levels.35

STATISTICAL ANALYSES
A voxel-based analysis was conducted using statistical parametric mapping (SPM99; FIL, Wellcome Department of Imaging Neuroscience, University College of London, London, England). For each subject, a mean image was made from the 3 123I-iomazenil emission scans scaled to the total volume of distribution (VT; milliliters per cubic centimeter). A mean iomazenil VT template was made from all study subjects and was spatially normalized to the SPECT blood flow template in SPM99 using a mask to occlude uncommon brain areas. Thereafter, each mean VT map from each subject was spatially normalized to the mean iomazenil SPECT VT template and smoothed using a gaussian kernel of 12 mm 12 mm 12 mm. Statistical analyses between comparison groups were conducted without global normalization. Overall effects were determined using an F threshold of 300 between 5 groups, including controls, smokers with alcohol dependence who were abstinent from alcohol for less than 1 week, nonsmokers with alcohol dependence who were abstinent from alcohol for less than 1 week, smokers with alcohol dependence who were abstinent from alcohol for less than 4 weeks, and nonsmokers with alcohol dependence who were abstinent from alcohol for less than 4 weeks. The t statistic images were thresholded to a minimum cluster size of 42 voxels, calculated based on the nominal resolution, ie, the full width at half maximum of a point source in water in the Picker Prism 3000XP SPECT camera. Betweengroup analyses were conducted comparing subjects in the control and alcohol-dependent groups. In the alcohol-dependent groups, the relationship between 123I-iomazenil VT and various clinical measures of alcohol dependence were explored. The anatomic location of the most significant voxel in clusters demonstrating statistical significance after corrections for multiple comparisons was determined after conversion of Montreal Neurological Institute stereotaxic coordinates to Talairach coordinates and submission of the results to Talairach Daemon.36,37 Region of interest data were analyzed using SAS version 8.2 (SAS Institute Inc, Cary, NC). Data were examined for normality using normal probability plots and Kolmogorov-Smirnov test statistics. Differences in VT between subjects in the control and alcohol-dependent groups and between smokers and nonsmokers were assessed using a mixed-effects regression model with 123 I-iomazenil VT as the dependent variable and with group (subjects with alcohol dependence or control subjects), smoking status (yes or no), region of interest, and interactions as fixed factors and unstructured variance-covariance matrix across regions. Nonsignificant interactions were dropped from the model for parsimony. Age was considered as a covariate but was not significant and was dropped from the model. Correlations with clinical variables for the patients were also assessed using mixed models with 123I-iomazenil VT as the dependent variable and smoking, region of interest, 1 clinical variable at a time, and all possible interactions as independent variables. Different slopes for smokers and nonsmokers were estimated to illustrate the relationship between significant clinical variables and the dependent variable. Statistical significance was considered at P =.05. Bonferroni

I-IOMAZENIL SPECT IMAGING AND MAGNETIC RESONANCE IMAGING

123 I-iomazenil was prepared as described previously.32 The average yield was a meanSD of 63.0%11.7% (n=57 preparations) and its radiochemical purity was a mean SD of 97.8%1.8%. All participants were pretreated with saturated potassium iodide to reduce thyroid uptake of 123I. 123I-iomazenil was administered by intravenous injection (71.32.1 MBq, 72.61.9 MBq, and 72.51.5 MBq for subjects with alcohol dependence at 1 week of abstinence, subjects with alcohol dependence at 4 weeks of abstinence, and control subjects, respectively) plus constant infusion (275.9 7.3 MBq/h, 280.1 7.7 MBq/h, and 278.25.1 MBq/h, respectively). The duration of infusion was 6.5 hours with a meanSD total injected dose of 221.23.1 MBq for subjects with alcohol dependence and 223.21.8 MBq for control subjects. Prior to imaging, 5 external fiducial markers containing 0.037 MBq of 123I were placed on the scalp to provide a common reference for coregistration with emission images. Simultaneous transmission and emission scans were acquired with a line source containing 740 MBq of cobalt 57 on a PRISM 3000XP 3-headed camera (Picker International, Cleveland, Ohio). Three consecutive 12-minute emission scans were acquired in continuous mode between 330 and 370 minutes of 123 I-iomazenil infusion. Three venous blood samples were collected at the midpoint of the SPECT scan (at 350 minutes) for measurement of free 123I-iomazenil.32 123I-iomazenil SPECT has 83% to 90% reproducibility.33 Magnetic resonance imaging was performed on a Signa 1.5T system (General Electric Co, Milwaukee, Wis). Axial images were acquired parallel to the anteroposterior commissural line with an echo time of 5 milliseconds; repetition time of 24 milliseconds; matrix 256 192; number of excitations of 1; field of view of 24 cm; and 124 contiguous slices with a thickness of 1.2 mm.

123

IMAGE ANALYSIS
Emission data from SPECT scans were reconstructed from counts acquired in the 123I photopeak (159 keV) with a 20% symmetric window using a Butterworth filter (power factor = 10, cutoff=0.24 cm). An attenuation map was reconstructed from the transmission and flood data and was used for nonuniform attenuation correction.34 The magnetic resonance image was coreg-

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Table 1. Demographics and Smoking Characteristics of Healthy Nonsmokers, Healthy Smokers, Nonsmokers With Alcohol Dependence, and Smokers With Alcohol Dependence*
Scan 1 (at 1 Week) Characteristic Age, y FTND score Cotinine, ng/mL MNWS HNS (n = 10) 35.9 7.5 0 50 0.5 1.0 HS (n = 5) 41.2 9.2 5.0 2.5 305.6 91.8 3.2 3.3 ANS (n = 8) 39.9 8.6 0 50 2.8 1.7 AS (n = 15) 40.9 8.1 5.7 2.7 185.1 122.0 10.9 6.7 Scan 2 (at 4 Weeks) ANS (n = 6) 40.8 9.9 0 50 6.4 5.1 AS (n = 14) 38.4 7.0 5.1 2.5 216.7 90.8 6.3 10.8

Abbreviations: ANS, nonsmokers with alcohol dependence; AS, smokers with alcohol dependence; FTND, Fagerstrom Test for Nicotine Dependence; HNS, healthy nonsmokers; HS, healthy smokers; MNWS, Minnesota Nicotine Withdrawal Scale. *Data are presented as mean SD. Eight of 10 HNS were never smokers; all ANS were never smokers. Value is approximate. Nicotine withdrawal was assessed using the MNWS on the day of the single-photon emission computed tomographic scan.

Table 2. Characteristics of Nonsmokers With Alcohol Dependence and Smokers With Alcohol Dependence*
Scan 1 (at 1 Week) Characteristic Paternal history of alcohol dependence, No. Days since last alcoholic drink consumed Age at onset, y Alcohol units in last month Peak CIWA score Alcohol dependence scale score Tiffany scale for craving score ANS (n = 8) 6 4.6 1.8 27.8 8.0 340.6 148.7 6.9 4.8 17.4 6.7 119.9 35.3 AS (n = 15) 9 5.0 2.9 25.2 11.3 490.7 170.4 4.5 2.9 18.9 7.0 127.1 65.0 Scan 2 (at 4 Weeks) ANS (n = 6) 4 26.7 2.8 26.5 9.0 381.2 135.6 8.2 5.0 17.2 5.6 107.3 38.6 AS (n = 14) 7 31.1 8.7 23.4 10.2 444.3 194.6 4.9 3.3 20.2 6.6 100.3 23.6

Abbreviations: ANS, nonsmokers with alcohol dependence; AS, smokers with alcohol dependence; CIWA, Clinical Institute Withdrawal Assessment. *Data are presented as mean SD unless otherwise indicated. Four smokers with alcohol dependence and 2 nonsmokers with alcohol dependence dropped out of study after scan 1. Three smokers with alcohol dependence participated in scan 2 but not in scan 1. One subjects family history was unknown.

correction was used to correct for multiple comparisons for post hoc region of interest analyses. RESULTS

CLINICAL POPULATION Twenty-three men with alcohol dependence, including 15 smokers and 8 nonsmokers, and 15 control men, including 5 smokers and 10 nonsmokers, were included in the clinical population (Table 1). Smokers in the alcoholdependent and control groups demonstrated similar Fagerstrom Test for Nicotine Dependence scores. Smokers with alcohol dependence tended to have lower plasma cotinine levels compared with control smokers (t17 =2, P =.06). Subjects with alcohol dependence reported smoking, on average, 1 pack of cigarettes per day, which was similar to control smokers; however, once they became inpatients where they had up to 4 escorted smoking breaks each day, they were not able to match their outpatient smoking frequency, as demonstrated by differences in plasma cotinine levels and the severity of nicotine withdrawal (Table 1). More than half of the men with alcohol dependence exhibited a paternal history of alcohol dependence, with a higher proportion of this family history in nonsmokers as compared with smokers. The average age
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at onset, the severity of alcohol dependence, and alcohol craving were similar between smokers and nonsmokers. An insignificant trend toward greater severity of alcohol withdrawal symptoms was noted in the nonsmokers as compared with smokers (Table 2). Smokers with alcohol dependence drank more alcohol than nonsmokers with alcohol dependence in the past month. I-IOMAZENIL UPTAKE AT 1 AND 4 WEEKS OF SOBRIETY There were no between-group differences in the injected dose, bolus-infusion ratio, and time of scan (data not shown). There was a trend difference in meanSD levels of total 123I-iomazenil: control nonsmokers, 0.180.04 kBq/ mL; control smokers, 0.160.03 kBq/mL; nonsmokers with alcohol dependence who were abstinent from alcohol for less than 1 week, 0.150.03 kBq/mL; smokers with alcohol dependence who were abstinent from alcohol for less than 1 week, 0.140.03 kBq/mL; nonsmokers with alcohol dependence who were abstinent from alcohol for less than 4 weeks, 0.160.05 kBq/mL; and smokers with alcohol dependence who were abstinent from alcohol for less than 4 weeks, 0.140.04 kBq/mL; where F2,31 =3.06 and P =.06. There was also a trend difference in meanSD levels of free 123I-iomazenil: control nonsmokers, 0.070.02
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kBq/mL; control smokers, 0.050.01 kBq/mL; nonsmokers with alcohol dependence who were abstinent from alcohol for less than 1 week, 0.050.01 kBq/mL; smokers with alcohol dependence who were abstinent from alcohol for less than 1 week, 0.050.01 kBq/mL; nonsmokers with alcohol dependence who were abstinent from alcohol for less than 4 weeks, 0.05 0.01 kBq/mL; and smokers with alcohol dependence who were abstinent from alcohol for less than 4 weeks, 0.050.01 kBq/mL; where F2,31 =5.30 and P =.01, with lower levels in subjects with alcohol dependence compared with levels in control subjects. The difference in 123I-iomazenil clearance is corrected for in the outcome measure, VT, which is equal to the total regional activity divided by the free 123I-iomazenil in blood. Plasma 123I-iomazenil levels were similar between smokers and nonsmokers. 123 I-iomazenil uptake was higher throughout the brain for subjects with alcohol dependence, but the degree of difference and significance varied in a region-dependent manner. A voxel-based analysis designed to determine if there was any group time interaction in a 1-way analysis of variance of alterations in 123I-iomazenil uptake between (1) control nonsmokers, n=10, plus control smokers, n=5 (123I-iomazenil VT uptake did not differ between control smokers and nonsmokers, and n=5 is too small for voxel-based analyses; therefore, control smokers and nonsmokers were pooled); (2) smokers with alcohol dependence who were abstinent from alcohol for less than 1 week (n = 15); (3) nonsmokers with alcohol dependence who were abstinent from alcohol for less than 1 week (n=8); (4) smokers with alcohol dependence who were abstinent from alcohol for less than 4 weeks; and (5) nonsmokers with alcohol dependence who were abstinent from alcohol for less than 4 weeks. This analysis demonstrated significant differences in left frontal inferior and middle gyrus, Brodmann areas (BAs) 11 and 47; right occipital lobe, BA 19; left and right temporal lobe, BA 21; and the cingulate gyrus, BA 31. The most significant cluster (with the highest F value) was noted in the frontal cortex. A plot of this cluster demonstrated higher 123I-iomazenil uptake in nonsmokers with alcohol dependence who were abstinent from alcohol for less than 1 week compared with smokers with alcohol dependence who were abstinent from alcohol for 1 week and control subjects. This effect appeared to normalize by 4 weeks of abstinence (Figure 1). Analysis of the t statistic maps comparing nonsmokers with alcohol dependence who were abstinent from alcohol for less than 1 week vs control subjects (n=15) indicated significantly higher 123I-iomazenil uptake in the frontal (BA 47), insular (BA 13), occipital (BAs 17 and 19), temporal (BA 21), and parietal (BA 40) cortices as an overall group (P =0; set-level) and as 6 individual clusters after corrections for multiple comparisons, with a P value range of .002 to .05 (Figure 2). The effects of smoking were also examined using a mixed-model analysis for 4 brain areas, including medial frontal cortex, anterior cingulate cortex, hippocampal-amygdala area, and cerebellum. There was significant group by smoking interaction (F1,34 =5.54; P =.02) with significant differences between control subjects and nonsmokers with alcohol dependence who were abstinent from alcohol for 1 week (28.4%, 29.5%, 31.6%, and 13.7% for medial frontal cortex, ante(REPRINTED) ARCH GEN PSYCHIATRY/ VOL 62, AUG 2005 881

rior cingulate cortex, hippocampal-amygdala area, and cerebellum, respectively; F1,34 = 6.24; P = .02), but not between control subjects and smokers with alcohol dependence who were abstinent for 1 week (14%, 14%, 6.2%, and 5.2% for medial frontal cortex, anterior cingulate cortex, hippocampal-amygdala area, and cerebellum, respectively; F1,34 = 0.81; P = .37) (Figure 3, Table 3). There were no significant differences between smokers in the control and alcohol-dependent groups and nonsmokers in these groups who were abstinent from alcohol for 4 weeks. There was a significant positive association between the number of days of abstinence from alcohol for nonsmokers with alcohol dependence (t19 =2.35, P = .03) but not for smokers with alcohol dependence (t19 =0.34, P =.70) (Table 4). Pearson product correlations demonstrated statistically significant correlation coefficients between days of abstinence from alcohol and 123 I-iomazenil uptake in medial frontal cortex and cerebellum (Figure 4), suggesting that GABAA-BZ receptors increased over the first week of abstinence in nonsmokers with alcohol dependence but not in smokers with alcohol dependence. Voxel-based comparisons of 123I-iomazenil uptake between subjects with alcohol dependence who were abstinent from alcohol for less than 1 week and for less than 4 weeks did not yield significant differences in gray matter brain areas (data not shown). Likewise, in the mixedmodel analysis, 123I-iomazenil VT was not significantly different between smokers and nonsmokers with alcohol dependence between 1 and 4 weeks of abstinence from alcohol. These findings are not consistent with the early findings in the F test analysis, most likely because of the small number of subjects in the alcohol-dependent nonsmoker groups. RELATIONSHIP BETWEEN 123I-IOMAZENIL UPTAKE AND CLINICAL FEATURES The relationship between regional 123I-iomazenil uptake and various clinical characteristics of the subjects with alcohol dependence were assessed (Figure 4 and Figure 5; Tables 2 and 4). A significant positive correlation with 123 I-iomazenil uptake in medial frontal cortex and cerebellum and the days since the last alcoholic drink was consumed was noted at 1 week of abstinence in nonsmokers with alcohol dependence but not in smokers with alcohol dependence. Voxel-based comparisons in subjects with alcohol dependence who were abstinent from alcohol for less than 1 week demonstrated significant positive correlation with the severity of alcohol withdrawal in the occipital lobe (BAs 17, 18, and 19) and the cerebellum, which was also demonstrated in the cerebellum region of interest analysis. There were no significant correlations with the other clinical variables.
COMMENT

The present study evaluated adaptive changes in GABAABZreceptor availability in subjects with alcohol dependence over the first month of sobriety using SPECT and 123 I-iomazenil. 123I-iomazenil is a potent competitive BZ antagonist with weak inverse agonist properties38,39 that has pharmacological specificity similar to flumazenil, a wellWWW.ARCHGENPSYCHIATRY.COM

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A
Contrast(s)
1

[18, 24, 15]

F Statistic D F Statistic

1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 1 2 3 4 5

Scan No.

400

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110 ANS 4 wk 100 AS <1 wk AS 4 wk

90 Control

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Figure 1. Voxel-based analysis using statistical parametric mapping (SPM99; FIL, Wellcome Department of Imaging Neuroscience, University College of London, London, England) of F test of all effects for iodine I 123labeled iomazenil regional distribution volume (VT; all F5,53300). A, The projection views illustrate the regions where 123I-iomazenil regional distribution volume is altered (set-level P value=0 for all clusters; height threshold F=300.00; extent threshold k=0 voxels) for the comparison illustrated in the design matrix, including group 1, all control subjects (10 nonsmokers and 5 smokers; scan numbers 1-15); group 2, smokers with alcohol dependence (AS) who were abstinent from alcohol for less than 1 week (n=15; scan numbers 16-31); group 3, nonsmokers with alcohol dependence (ANS) who were abstinent from alcohol for less than 1 week (n=8; scan numbers 32-38); group 4, AS who were abstinent from alcohol for less than 4 weeks (n=14; scan numbers 39-52); and group 5, ANS who were abstinent from alcohol for less than 4 weeks (n=6; scan numbers 53-58). B, Plot of the activity (fitted and adjusted responses for all effects) at the voxel with the highest F value, suggesting that the ANS who have been abstinent from alcohol for less than 1 week have higher uptake compared with all other groups. C, The region localization of significant clusters on mean 123I-iomazenil regional distribution volume maps of all study participants. D, Representative magnetic resonance image from a single subject.

known, structurally related BZ antagonist,39-43 and Ro4513, a partial inverse agonist. 123I-iomazenil binds to all 1 through 6 subtypes and thus is a measure of all GABAA receptors in the brain. The present findings demonstrate higher 123I-iomazenil binding to GABAA-BZ receptors in the parietal, frontal, cingulate, temporal, insular, and occipital cortices of nonsmokers with alcohol dependence at 1 week of abstinence from alcohol but not of smokers with alcohol dependence at 1 week of abstinence from alcohol, as compared with the control group. The GABAA-BZreceptor availability correlated with the severity of alcohol withdrawal in all of the subjects with
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alcohol dependence and correlated positively with the number of days since the last alcoholic drink was consumed by the nonsmokers with alcohol dependence but not the smokers with alcohol dependence. The timedependent changes in GABAA-BZreceptor availability are supported by an earlier study17 that demonstrated a timedependent change in response to BZ challenge in orbitofrontal and cingulate cortices in subjects with alcoholism who were abstinent from alcohol for 2 to 3 weeks and for 6 to 8 weeks. Collectively, these findings suggest that both time since consuming the last alcoholic drink and smoking status are critical variables that need
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A Contrast(s)
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[18, 21, 15]

t Statistic D t Statistic

1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 1 2 3 4 5

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Figure 2. Voxel-based analysis (t test) demonstrating higher iodine I 123labeled iomazenil uptake in nonsmokers with alcohol dependence who were abstinent from alcohol for less than 1 week compared with control subjects. A, The t statistic map (t53; height threshold t = 3.25; extent threshold k = 42 voxels) illustrates brain regions with higher 123I-iomazenil regional distribution volume in nonsmokers with alcohol dependence who were abstinent from alcohol for less than 1 week (n = 8) compared with control subjects (n=15), as illustrated in the design matrix (group 3group 1). (For explanation of groups and scan numbers, see Figure 1A legend.) B, Scatter plot illustrates the regional distribution volume (VT) centered around the mean at the voxel with the highest F value for controls (1 on the x-axis) and nonsmokers with alcohol dependence (3 on the x-axis). C, The region localization of significant clusters on mean 123 I-iomazenil regional distribution volume maps of all study participants. D, Representative magnetic resonance image from a single subject.

to be taken into account when studying the adaptations in GABAA-BZreceptor expression throughout recovery from alcohol dependence. Increased GABAA-BZreceptor expression, as well as augmented GABA-mediated enhancement of BZ binding, has been observed in the superior frontal cortex of postmortem human brain from subjects with alcohol dependence who were without complications.18,19,44 Since BZ binding is differentially sensitive to subunits,45 the increase may be a consequence of subunit changes that occur in response to abrupt withdrawal after chronic alcohol consumption.46 In postmortem frontal cortex from humans with alcoholism, 1 and 3 messenger RNA (mRNA) is enhanced whereas 2, 3, and 4 mRNA is not significantly altered.47,48 In rats chronically treated with
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ethanol, levels of cortical 1, 2, and 5 mRNA and 1, 2, and 3 polypeptides are decreased whereas 4 mRNA levels are increased.49-53 During withdrawal, 1 and 4 levels rapidly normalize.54,55 The decrease in 1 subunit levels appears to be owing to ethanol-induced internalization of cortical 1-GABAA receptors56 that results in decreased cell surface and increased intracellular GABAA-BZ receptors. It is not clear whether iomazenil labels cell surface or intracellular receptors or binds to both receptor populations. If iomazenil labels both populations, then the observed increase reflects increased internalized or intracellular receptors in addition to decreased cell surface receptors, which would have an overall net effect of decreased GABAA-BZreceptor function during early sobriety.
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A
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Figure 3. Scatterplots illustrating individual iodine I 123labeled iomazenil regional distribution volume (VT) values for region of interest analyses. Regional distribution volume (regional activity/free plasma parent) determined from the region of interest analyses for each individual subject is illustrated for the medial frontal cortex (A), anterior cingulate cortex (B), hippocampal-amygdala area (C), and cerebellum (D) for the healthy nonsmokers (HNS), healthy smokers (HS), nonsmokers with alcohol dependence (ANS) who were abstinent from alcohol for less than 1 week, smokers with alcohol dependence (AS) who were abstinent from alcohol for less than 1 week, ANS who were abstinent from alcohol for less than 4 weeks, and AS who were abstinent from alcohol for less than 4 weeks. The mixed-regression analysis suggested that when analyzed as a group, the ANS who were abstinent from alcohol for less than 1 week were greater in the total volume of distribution than HNS and HS, and when analyzed individually, this comparison was significant only in the hippocampal-amygdala area.

Table 3. Benzodiazepine-Receptor Distribution Volume in Different Brain Regions of Subjects in Control and Alcohol-Dependent Groups*
Medial Frontal Cortex Controls Healthy men (n = 15) Smokers (n = 5) Nonsmokers (n = 10) After 1 wk of abstinence from alcohol Men with alcohol dependence (n = 23) Smokers (n = 15) Nonsmokers (n = 8) After 4 wk of abstinence from alcohol Men with alcohol dependence (n = 20) Smokers (n = 14) Nonsmokers (n = 6) 104.8 18.8 107.6 19.5 99.4 14.2 118.4 24.6 113.4 23.6 127.6 25.4 112.1 22.2 110.3 24.2 116.1 18.2 Anterior Cingulate Cortex 110.4 22.0 115.6 23.5 103.0 15.4 123.0 25.1 117.4 22.3 133.4 28.3 118.6 25.3 116.9 22.7 122.3 32.7 Hippocampal-Amygdala Area 74.3 11.2 77.5 8.3 70.8 10.9 81.4 19.0 75.2 13.7 93.2 22.7 78.4 14.6 76.5 15.3 83.0 13.0

Cerebellum 92.5 19.9 90.9 25.8 89.8 15.1 97.2 19. 94.5 21.0 102.1 15.2 95.8 16.5 95.6 17.5 96.2 15.6

*Data are presented as mean SD regional distribution volumes. P value of .009 not corrected for multiple comparisons when the mixed-model analysis was restricted to the comparison between subjects with alcohol dependence who were abstinent from alcohol for less than 1 week and healthy control subjects.

EFFECTS OF SMOKING AND RELEVANCE TO GABAA-BZ RECEPTORS The up-regulation in 123I-iomazenil uptake and the correlation with days since consuming the last alcoholic drink observed in nonsmokers with alcohol dependence were attenuated in smokers, suggesting that smoking suppressed the increase in levels of GABAA-BZ receptors during early sobriety. This finding is of great importance given the high prevalence (up to 90%) of smoking among sub(REPRINTED) ARCH GEN PSYCHIATRY/ VOL 62, AUG 2005 884

jects with alcohol dependence. While smokers tend to suffer from more severe alcohol dependence,57-59 they also report feeling less intoxicated on alcohol challenge.60 In the present study, there was a trend for smokers with alcohol dependence to report fewer alcohol withdrawal symptoms, suggesting that smoking may reduce the severity of withdrawal. In support of this, mice treated long-term with ethanol develop a greater and long-lasting response to inverse agonists, suggesting that BZ inverse agonists may antagonize ethanol intoxication.61,62 These findings, comWWW.ARCHGENPSYCHIATRY.COM

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Table 4. Correlation of Iodine I 123Labeled Iomazenil Uptake in Subjects With Alcohol Dependence Who Were Abstinent From Alcohol for Less Than 1 Week
Smokers With Alcohol Dependence (n = 15) Correlation Coefficient (r ) No. of days since last drink Medial frontal cortex Anterior cingulate cortex Hippocampal-amygdala area Cerebellum CIWA Medial frontal cortex Anterior cingulate cortex Hippocampal-amygdala area Cerebellum 0.14 0.15 0.04 0.03 0.04 0.06 0.17 0.10 P Value* .61 .60 .89 .91 .89 .83 .54 .73 Nonsmokers With Alcohol Dependence (n = 8) Correlation Coefficient (r ) 0.72 0.65 0.60 0.93 0.59 0.57 0.48 0.71 P Value* .05 .08 .12 .001 .12 .14 .23 .05

Abbreviation: CIWA, Clinical Institute Withdrawal Assessment. *P values are not corrected for multiple comparisons. When the Bonferroni correction for multiple comparisons is applied, statistical significance is observed only in the cerebellum for the number of days since the last alcoholic drink was consumed (P = .01).

bined with the observation for a tendency for 123I-iomazenil to correlate with the reported severity of withdrawal in the nonsmokers with alcohol dependence but not in smokers with alcohol dependence, suggest that the greater the adaptive increase in GABAA-BZreceptor expression is, the more severe the withdrawal symptoms are. Thus, it would appear that smoking might prevent GABAAreceptor adaptations associated with alcohol dependence and withdrawal. Furthermore, these findings predict that smokers with alcohol dependence should have greater success with abstaining from alcohol should they continue to smoke during acute withdrawal. Moderate smokers tend to increase the intensity of smoking during ethanol detoxification to suppress ethanol withdrawal symptoms.63 Several constituents of tobacco smoke may play a role in the impact of smoking. Nicotine directly stimulates GABA neuronal activity and may, therefore, suppress alcohol withdrawal symptoms.64 Cigarette smoke also contains -carbolines that have been measured in the plasma from smokers in the control and alcohol-dependent groups.65 The -carbolines are well known as monoamine oxidase inhibitors but have also been suggested to be GABAA-BZ receptor inverse agonists and to antagonize some of the effects of ethanol. There has been some debate about whether the -carbolines reach sufficient levels in the brain to have pharmacological effects.66,67 They do achieve sufficient levels to inhibit monoamine oxidase enzymes,68-71 but they have a 2- to 10-fold lower affinity for binding to GABAA-BZ receptors as compared with monoamine oxidase.66,67,72-74 Definitive conclusions about the role of the -carbolines on GABAA-BZreceptor availability in the present study without plasma measures are speculative, and this should be addressed in future studies. Additionally, other compounds in tobacco smoke or other receptor mechanisms may be responsible for the opposing effects of tobacco smoke on alcohol withdrawal symptoms. RELATIONSHIP OF SMOKING TO CHANGES IN CORTICAL GABA DURING EARLY RECOVERY Interpretation of the current study in light of concurrent cortical GABA level measurements made in the same
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A
200 150

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200 150 100 mFC CB 0 2 4 6 8 10 50 0 0 2 4 6 8 10 mFC CB

VT, mL/mL

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Days Since Last Drink

Days Since Last Drink

Figure 4. Graph illustrating the correlation between iodine I 123labeled iomazenil uptake and the number of days since the last alcoholic drink was consumed. The correlation between the number of days since consuming the last alcoholic drink and individual 123I-iomazenil regional distribution volume (VT) values were plotted for the medial frontal cortex (mFC) and cerebellum (CB) for nonsmokers with alcohol dependence who were abstinent from alcohol for less than 1 week of abstinence (A) and smokers with alcohol dependence with less than 1 week of abstinence from alcohol (B). Note the significant correlation between 123I-iomazenil VT and the number of days since consuming the last alcoholic drink in the nonsmoker group and the lack of correlation in the smoker group.

subjects may shed light on the functional implications of the time-dependent changes in 123I-iomazenil observed in subjects in this study.75 The GABA levels are modulated by GABAA-BZ receptors to maintain GABAergic neurotransmission. For example, clonazepam produces a substantial decrease in cortical GABA levels measured using 1H-magnetic resonance spectroscopy in healthy human subjects.76 Thus, it is possible that a shift from 4 or 6 BZ-insensitive, inverse agonistsensitive GABAA receptors to 1 BZ-sensitive GABAA receptors during the recovery from alcohol dependence would be marked by a decline in cortical GABA levels. In fact, nonsmokers with alcohol dependence in this study exhibited normal cortical GABA levels in the first week of abstinence from alcohol that declined over the first month of sobriety75 and remained low over the next 5 months whereas smokers with alcohol dependence demonstrated low cortical GABA levels that did not vary between 1 and 4 weeks of
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A
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Figure 5. Voxel-based analysis demonstrating the correlation between iodine I 123labeled iomazenil regional distribution volume (VT) and the severity of alcohol withdrawal. Voxel-based correlation between the severity of alcohol withdrawal was determined using the Clinical Institute Withdrawal Assessment (CIWA) and 123 I-iomazenil VT values for all subjects with alcohol dependence (n=23) with less than 1 week of abstinence from alcohol. A, The t statistic map (t16; height threshold t =3.69; extent threshold k=42 voxels). Design matrix shows relationship between 7 clinical variables, where group 4 is the peak CIWA score; the scan numbers refer to the 23 smokers and nonsmokers with alcohol dependence who were abstinent from alcohol for less than 1 week. B, Plot of the activity (fitted and adjusted responses) at less than 1 week of abstinence from alcohol; distribution volumes (VT) are centered around the mean at the highest t statistic value. C, The region localization of significant clusters on mean 123I-iomazenil VT maps of all study participants. D, Representative magnetic resonance image from a single subject.

abstinence.75,77 In nondrinkers, cortical GABA levels do not vary between men who do and who do not smoke.78 The effects of smoking on GABAA-BZreceptor availability have yet to be evaluated. The relationship of the subunit substitutions during alcohol dependence and withdrawal to GABAA-receptor function is not clear. However, there is a wealth of data demonstrating decreased GABAAreceptor function in response to chronic ethanol use that may occur owing to increased inverse agonistsensitive 4 subunits that increase the sensitivity to inverse agonists that would reduce GABAA-receptor function. With sobriety, the restoration of BZ-sensitive, inverse agonist insensitive GABAA receptors may alleviate a GABAA-BZ receptor functional deficit and may contribute to the decline in cortical GABAA binding and GABA levels associated
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with recovery. It is not yet clear whether the reductions in GABA levels, 123I-iomazenil uptake, and lorazepam response that may persist with extended sobriety reflect a persisting consequence of alcohol dependence, alcohol dependencerelated neurotoxicity, or a preexisting trait.79 IMPLICATIONS FOR TREATMENT OF ALCOHOL WITHDRAWAL The current data suggest that GABAA-receptor adaptations may contribute to acute ethanol withdrawal and to the recovery from alcohol dependence. While BZ treatments are the predominant detoxification strategy, a growing number of alternative approaches are being explored, including anticonvulsant medications and N-methyl-DWWW.ARCHGENPSYCHIATRY.COM

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aspartate glutamate receptor antagonists.80,81 The current data raise the possibility that treatments that accelerate the normalization of GABAA-receptor populations may increase the rate of recovery whereas treatments that have ethanol-like effects on GABAA-receptor populations may delay recovery. The effects of these detoxification strategies on alcohol-related adaptations in human GABAAreceptor populations are currently unknown. However, there is growing interest in the possibility that these treatments might avoid the negative effects of BZ-assisted detoxification on the initiation of abstinence in patients who have completed acute detoxification.80 In this regard, it is possible that substances in tobacco smoke, such as BZ inverse agonist -carbolines1 or nicotine, may provide clues to novel pharmacotherapeutic approaches to alcohol dependence that might prevent or treat acute withdrawal symptoms and promote the initiation and maintenance of sobriety. Submitted for Publication: July 30, 2004; final revision received January 10, 2005; accepted January 13, 2005. Author Affiliations: Yale University School of Medicine, New Haven, Conn (Drs Staley, Gottschalk, Petrakis, Gueorguieva, OMalley, Baldwin, Jatlow, Verhoeff, Perry, van Dyck, Seibyl, Innis, and Krystal, Mr Weinzimmer, and Mss Frohlich and Ruff); Veterans Affairs Connecticut Healthcare System, West Haven, Conn (Drs Staley, Petrakis, Baldwin, Verhoeff, Perry, Innis, and Krystal, Mr Weinzimmer, and Mss Frohlich and Ruff ); and Institute for Neurodegenerative Disorders, New Haven (Dr Seibyl). Dr Baldwin is now with the Department of Radiology, Vanderbilt University, Nashville, Tenn, Dr Verhoeff is with the University of Toronto, Toronto, Ontario, and Dr Innis is with the Molecular Imaging Branch, National Institute of Mental Health, Bethesda, Md. Correspondence: Julie K. Staley, PhD, Department of Psychiatry, Yale University School of Medicine & VACHS 116A2, 950 Campbell Ave, West Haven, CT 06516 (julie .staley@yale.edu). Funding/Support: This work was supported by the Dana Foundation, New York, NY, the Veterans Affairs Mental Illness Research, Education, and Clinical Center for Dual Diagnosis, and the Veterans Affairs Alcohol Research Center, West Haven, Conn, and grants K01AA00288, K05 AA014715-01, RO1 AA1132, and P50 DA13334 from the National Institutes of Health, Bethesda, Md. Previous Presentation: These findings have been presented in part at the Ninth Annual Meeting of the Society on Research on Nicotine and Tobacco; February 19-22, 2003; New Orleans, La; the Annual Meeting of the Society for Neuroscience; November 2002; Orlando, Fla; the Congreso Internacional las Neurosciencias y las Adicciones: Nuevos Desarrollos, Nuevas Esperanzas; June 2002; Mexico City, Mexico; the 25th Annual Meeting of the Research Society on Alcoholism; June 2002; San Francisco, Calif; the 26th Annual Meeting of the Research Society on Alcoholism; June 2003; Miami, Fla; and the 42nd Annual Meeting of the American College for Neuropsychopharmacology; December 2003; San Juan, Puerto Rico. Acknowledgment: We thank Louis Amici and Nina Sheung for their expert technical assistance in the synthesis and metabolite analyses of 123I-iomazenil.
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72. 73. 74. 75.

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